Summary The three-dimensional organization of the genome is critical for regulation of gene expression, maintenance of genome stability and chromosome inheritance. Over the last several years there has been a tremendous increase in our knowledge of the spatial arrangements of chromosomes, and this is leading to insights into the molecular mechanisms that regulate genes, and how defects in genome folding can lead to human disease. We have developed powerful molecular and genomic technologies based on chromosome conformation capture (3C, 5C, Hi-C) to probe the three-dimensional structure of chromosomes. As cells go through the cell division cycle chromosomes alternate between two entirely different spatial conformations. We and others have used 3C-based assays to determine the structure of the human genome in interphase and in metaphase. In interphase the genome is composed of several different types of chromosomal domains, while within these domains genes are regulated by specific looping interactions between genes and their regulatory elements. A different structure is observed in mitotic cells, when chromosomes become highly compacted. We discovered that in mitosis chromosomes fold as linear arrays of consecutive chromatin loops. We have delineated a series of folding intermediates that show how the interphase conformation is converted into the metaphase state. These intermediates include extended linear loop arrays in prophase and more compacted helical arrays of nested loops in prometaphase. These studies lead to important new questions that we aim to address. First, it is not known in genomic detail how during prophase the interphase state is erased, chromosomes form initial loop arrays and sister chromatids become separated. Second, very little is known about the molecular machines that fold chromosomes. We propose innovative new strategies to identify new components of these machines that act during mitosis and interphase. Third, we hypothesize that these machines act through specific cis-elements that determine how and where they get loaded onto chromosomes, move to new sites and accumulate at yet other sites. We will identify and characterize these DNA elements that encode how the genome folds. Our proposed studies will uncover how the genome folds, unfolds and refolds.